The goal of this project is to investigate molecular alterations in cell cycle control during neoplastic transformation. In one set of studies, cell cycle control in v-mos-transformed NIH3t3 cells is being investigated. Following removal of serum, these cells arrest in early G1 phase of the cell cycle and are unable to exit into quiescence. These G1 arrested cells express abnormally high levels and/or have abnormal kinase activity associated with p34cdc2, p33cdk2, cyclin D1, cyclin E, and cyclin A. When expression was induced in serum straved NIH3T3 cells containing constructs expressing either mos, ras, or MEK under the regulation of the sheep metallothionein promoter, mos expression resulted in increased expression of p34cdc2 that did not correlate with activation of MAP kinase. In fact, v-mos-transformed cell were found to be refractory to MAP kinase activation, suggesting possible alteration of protein phosphates in these cells. In other studies, the molecular mechanism of the G2 cell cycle checkpoint response to ionizing radiation (IR) is being analyzed in normal human fibroblasts and in fibroblasts that lack normal function of p53, pRB, or the ataxia telangiectasia (AT) cancer susceptibility gene product. Normal human fibroblasts respond to exposure to IR by rapidly delaying entry into mitosis with an associated strong inhibition of p34cdc2/cyclinB protein kinase activity. AT fibroblasts exposed to IR show little delay of entry into mitosis or inhibition of kinase activity. The rapid G2 checkpoint response to IR does not require p53, pRB, or p21 function. However, lack of p53 resulted in a progressively increasing proportion of cells evading the G2 checkpoint following irradiation that strongly correlated (R2=0.94) with the proportions of cells with aberrant metaphase chromosomes.